Analysis Of The Causing Mutation And Research On X-linked Gene Of Leber's Hereditary Optic Neuropathy Families

Objective:1.To establish the standard system to collect and preserve the familial genetic resource of Leber's hereditary optic neuropathy(LHON) families and isolated cases.2.To analyze the clinical and genetic features of the NO.HN001,HN002 and HN003 families.And to find the pathogenic mutation locus of these families.3.To find the X-linked modifier gene of LHON.And to explain the phenomenon of male predominance of LHON.Method:1.LHON probands and isolated cases were identified through related optic check-up.Their clinical features,family information were analyzed and they were eventrally identified as LHON patients after screening the causing LHON mutation.Collecting and preserving LHON genetic resource included family members'signing the information consent,doctors'filling out LHON questionnaire,extracting genomic DNA from peripheral blood,numbering DNA samples and preserving genomic DNA.It included paper-based record management system and electronic record management system to establish LHON genetic resource system.2.The mitochondria DNA mutation at nucleotide position(NP) 11778,3460 and 14484 were screened in the NO.HN001,HN002 and HN003 families using polymerase chain reaction(PCR),direct sequencing,restriction endonucleases. Compared the mtDNA sequence with revised Cambridge reference sequence and found the changed nucleotide locus.Looked up these changed locus in the international professional hereditary network in order to identify the causing mtDNA mutation of the NO.HN001,HN002 and HN003 families.3,Fluorecence-based genescan with microsatellite markers was carried out in the NO.HN001 family using 4 microsatellite markers(DXS8090,DXS984, DXS7,DXS7487) that had been reported to be linked.Haplotype analysis was performed and the data was analysed using Linkage Analysis Package 5.0 in order to find out whether X-linker modifer gene was linked to the 4 markers or there was no X-linker gene at all.Results:1.The system of collecting and preserving genetic resource of LHON was standard and scientific.It was in line with the principles of genetic resource collection,the international and domestic requirements.2.G11778A mutation was found in the NO.HN001,HN002,HN003 families. 4 additional changed loci which included G11719A,G14476A,C14766T and T14783C were detected in the NO.HN001 family.3.Fluorecence-based genescan and haplotype analysis showed no cosegregation of haplotype with 4 linked gene markers(DXS8090,DXS984, DXS7,DXS7487).LOD score from linkage analysis were negative for all 4 markers.Conclusion:1.Establishment of the system to collect and preserve LHON genetic resource ensured that this study was standard and benefited further study.It made our following results of molecular study and gene screening true and reliable.2.The NO.HN001 HN002 and HN003 families were LHON families. G11778A was the causing mutation.G14476A was a new kind of polymorphism of mtDNA whose corresponding coding protein didn't change which represented that it was a nonsense variation.This polymorphism existed in 3%of healthy people.3.there was no X-linked modifier gene around the 4 gene markers(DXS8090, DXS984,DXS7,DXS7487).Beause that the hereditary pattern of the modifier gene might be not X-chromosome recessive,or that Chinese and Asian people had different modifier genes compared with foreigners.The existance of the X-linked gene needed further study.